This invention relates to the use of a single, substantially pure, water soluble quaternary azo dye for the cytological preparation of a fixed biopsy specimen derived from human blood, bone marrow, lymph nodes and other specimen of hematopoietic origin. The use of a water soluble quaternary azo dye (identified in the Colour Index as Basic Blue 41) in accordance with this invention, is an advance over the prior art (Romanowsky and Malachowski) wherein a mixture of dyes were used for staining biopsy specimens. The stained cells of hematopoietic origin in accordance with this invention have excellent Colour stability and are remarkably clear with respect to cellular detail and brilliance of cell structure.
The use of the water soluble quaternary azo dye of this invention is an improvement over the above-mentioned Romanowsky stains even though the panoptic mixture of dyes are still the basis of morphological hematology and are presently being used for the identification of cells of hematopoietic origin including human blood cells, bone marrow cells, and lymph node cells.
Ehrlich introduced the use of dyes to effect or enhance cell differentiation in human biopsy specimens particularly blood cells. Ehrlich's dyes were superceded, however, by the use of mixtures of dyes identified as Romanowsky dyes which have been modified to include mixtures such as methylene blue, modified methylene blues, eosins, azures and methylene violet. These mixtures of dyes have been generally classified as panoptic stains because of the wide range and broad spectra of hues and chroma produced when reacted with a fixed biological specimen such as human blood.
As early as 1891, Romanowsky and Malachowski developed mixtures of polychromed methylene blue, azure and methylene violet. Other contributors including names such as Unna (1891), Nocht (1898), Jenner (1899), Leishman (1901), Wright (1902), May-Grunwald and Giemsa (1902), MacNeal (1906) and Lillie (1943) made contributions to what is generally referred to as Romanowsky derived panoptic dye mixtures. The various mixtures developed by the foregoing contributors to advance the state of the art are illustrated in Table I:
TABLE I ______________________________________ Meth- Polychromed Meth- ylene Methylene ylene Eosin Blue Blue Azure(s) Violet ______________________________________ Romanowsky X X X (1981) Malachowski X X X (1891) Jenner (1899) X X Leishman X X (1901) Wright (1902) X X Giemsa X X (1902-04) MacNeal X X X X (1906) ______________________________________
The state of the art, however, as presently known fails to disclose the use of a single, substantially pure, commercially available azo dye capable of panoptically and metachromatically staining fixed biopsy specimens comprising blood cells, bone marrow cells, or lymph node cells which permit the differentiation, enumeration and identification of the individual cells including leukocytes, and the subpopulations of lymphocytes, megakaryocytes, etc., or any of the abnormal or malignant cells.
The diagnosis of hematological disorders has been achieved, for the most part, by enumeration and identification of formed elements of the peripheral blood and bone marrow. The basis of hematological diagnosis includes the use of light microscopic examination of a panoptically stained specimen of blood cells, lymph node cells, or bone marrow cells. This type of examination provides information sufficient to make a diagnosis which parallels the developments of panoptic light microscopy. Cytochemical stains have been developed to identify cell types more precisely than was possible by using panoptic stains. Cytochemistry represents biochemistry in a microscopic and submicroscopic level. When applied to cells and tissue, cytochemical stains identify enzymes, substrates and organelles. As biochemical probes, cytochemical stains often provide valuable insights regarding aberrations of cellular metabolism. Moreover, cytochemical stains can be selective for one cell type compared to another and therefore such stains have been used in various diagnostic processes especially in making distinctions between various cytological types of acute leukemias and preleukemic disorders.
Concurrent with the development of the synthetic organic dyes, various investigators experimented with supravital stains by adding these dyes to freshly obtained samples of blood or suspension of cells. It was rapidly ascertained that some of the cells were stained with one or more of the dyes whereas other cells were not. Subsequent to the development of supravital staining of blood cells, Ehrlich found the need for a more stable, permanent preparation of blood cells that could be examined under the microscope. Recognizing the difficulties in cell identification in viewing suspensions of unstained cells, Ehrlich devised a stain composed of orange G, acid fuschin and methyl green. On the basis of differential coloration of leukocytes with a mixture of dyes, Ehrlich identified and named most of the blood leukocytes known today. Ehrlich's contribution was remarkable in that by using a plurality of dyes he was able to detect the difference in colors that were distinctive for various cell types. For example, those cells whose granules showed affinity for eosin were called eosinophils. Recognizing that some cells stained differently than the color of the dye in solution, the term metachromasia was popularized and applied to the granules of mast cells. At present, staining techniques form the basis of modern morphological hematology and the nomenclature of various cell types.
Early in the history of the morphologic and cytochemical diagnosis of blood disorders, it was appreciated that examination of only panoptically stained specimens of blood or bone marrow sometimes was not sufficient to make a diagnosis. While some investigators were popularizing supravital stains, others were describing cytochemical stains for blood cells that could be used on dried, fixed preparations of blood or bone marrow. It became apparent that there were some blood cells that had peroxidase activity while other cells did not. The peroxidase test was the first stain that reliably distinguished between granulocytic cells which contain activitiy of peroxidase and lymphoid cells which did not contain peroxidase activity. The peroxidase stains with chromophoric modifications and increased use in immunology remain one of the most useful stains in the cytochemistry of blood cells and is the basis for one of the current automated leukocyte differential counting instruments.
As a staining technique, however, cytochemistry has limitations with respect to age of sample, type of fixative, pH, presence or absence of heavy metal cations, deterioration of the substrate, time and temperature of the staining reaction, etc. These are all variables that affect the cytochemical stain. In addition, any impurities in the organic dyestuff as well as variability in the composition of the dye stuff causes alterations in the staining reaction. However, by using cytochemical stains, it is possible to identify the presence or absence of substances in one cell type contrasted to another or any increase or decrease in the quantity of such substance in those cell types. Quantitatively, these differences assume a diagnostic importance when they reflect differences in one cell type compared to another, and in normal cells compared to abnormal or pathological blood cells.
In several instances, specific diseases have cytochemical profiles that complement the traditional microscopic examination of panoptically stained preparations. There are a variety of hematologic disorders wherein cytochemical tests have diagnostic value. Complementing the conventional light microscopy of panoptically stained specimens of blood or bone marrow, cytochemical stains have improved the precision of hematological diagnosis with the recognition that these stains can reveal properties that are distinctive for one cell type compared to another. Cytochemical stains have found increased application in the study of blood, lymph node and bone marrow specimens. For the most part, these stains detect increased or decreased amounts of an enzyme or a metabolite that reflect the pathophysiological condition of a disordered cell. While the exact mechanism or chemistry responsible for the production of the cytochemical abnormalties are unknown, many of these abnormalities are sufficiently distinctive to make them useful diagnositcally. As a diagnostic tool for cellular hematology, cytochemistry represents a rapid and inexpensive method to distinguish one cell type from another on the basis of characteristic properties. With advances in dye chemistry and the automation of leukocyte counting, one can anticipate further improvements in the cytochemistry of blood cells and the precision of hematological diagnosis in the future. Discussions of cytochemical stains can be found in New Cytochemical Stains for Blood and Bone Marrow Cells and Cystobiology of Leukemias and Lymphomias, by L. Kass, M.D., Raven Press, New York, N.Y., Pages 161-177, 1985, and Lawrence Kass, M.D. Leukemia Cytology and Cytochemistry, published by J. B. Lippincott, Philadelphia, 1982.
Accordingly, it is an object of this invention to provide a single, substantially pure, water soluble azo dye for use in staining fixed cells of hematopoietic origin.
It is another object of this invention to provide cells of hematopoietic origin fixed and subsequently stained with a water soluble quaternary azo dye to obtain stained cells having individual color characteristics which permit the differentiation, identification and enumeration thereof.
It is a further object of this invention to provide a method of staining a plurality of cells of hematopoietic origin in a fixative to obtain stained cells having individual color characteristics which permit the differentiation, identification and enumeration by means of an instrument. These and other objects will be apparent from a further and more detailed description of the invention as follows.